S257: CAR T CELLS AND THEIR IMMUNE ENVIRONMENT SHAPE DISTINCT IMMUNE PROFILES IN RESPONSE AND TOXICITY IN B CELL LYMPHOMA PATIENTS
Authors: Stefanie Kreutmair, Maximilian Schaefer, Sebastian Stolz, Carla Merten, Can Ulutekin, Tobias Wertheimer, Florian Ingelfinger, Susanne Unger, Chiara Alberti, Aakriti Sethi, Markus Manz, Thorsten Zenz, Antonia Müller, Corinne Widmer, Burkhard Becher
Journal: HemaSphere (2023)

Background: Genetically engineered CD19-redirected chimeric antigen receptor T (CAR T) cells represent a breakthrough immunotherapy for B cell malignancies, but response rates are variable and some patients face life-threatening side effects such as cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). The impact of the patient’s immune landscape as a whole -consisting of CAR T cells and non-CAR T immune cells- on patients’ outcome is largely unknown. Furthermore, reliable biomarkers to predict response and toxicity are lacking. Aims: The aim is to decode the crosstalk of CAR T cells and their immune environment, link stratifying immune profiles to the patient’s outcome to get insights into underlying immunopathological mechanisms, and extract predictive biomarkers. Methods: We applied a systems immunology approach based on longitudinal (day -1 to 104 after CAR T infusion), high-dimensional single-cell cytometry and algorithm-guided computational analysis of 47 PB samples from 19 lymphoma patients, together with 6 healthy controls (HC). The generated immune map, based on 69 parameters and 2078 extracted immune features, was enriched with numerous clinical parameters, including response and toxicity data of a follow-up period ≥315 days. Results: This proteomics approach revealed a lymphoma patient-specific, heterogeneous CAR T cell population, which appeared to be only a minor fraction of cytokine producing T cells. Most of the identified 13 CAR T subsets, such as γδT cells and CD4 T regulatory cells, appeared in each patient sample, with a bias towards CD8 CAR T cells at early timepoints and dominating CD4 CAR T subsets later on. When stratifying the overall immune profile at the time of CAR T cell expansion and toxicities (day 9-21), immune alterations associated with response to CAR T therapy (best overall response complete remission) were primarily found in the non-CAR T immune compartments, while CRS- and ICANS-associated signatures affected predominantly the CAR T population. In particular, both CRS and ICANS were linked to a dysbalance in the CD226/TIGIT-axis, with high expression of the co-stimulatory receptor CD226 in CD4 and CD8 CAR T cells, whereas the co-inhibitory and competing receptor TIGIT together with its regulator EOMES demonstrated low expression. Suggestive for biological relevance, expression levels of CD226, TIGIT and EOMES strongly correlated with maximum clinical scores of CRS and ICANS. Lower expression of the immune checkpoint CTLA-4 was detected in both CAR+ and CAR- T cell subsets, indicating immunopathological signatures in CRS and ICANS are based on imbalanced activating/inhibitory receptor profiles. Focusing on the non-CAR T immune compartment, signs of exhaustion in T cells (TIM3, LAG3) and activation in NK cells (CD244) were linked to ICANS. By contrast, primarily T cell-associated immune features were detected in CRS, pointing to NK cells playing a minor role in this condition. Supportive for a pivotal role of the immune environment, stratifying immune signatures associated with later patient outcomes were already detected pre-CAR-T-infusion and resulted in significant and high accuracy of outcome prediction – of high clinical relevance for patient selection and management. Summary/Conclusion: Our systems immunology approach allowed us to ultimately define the CAR T specific immune landscape. These results could serve as predictive biomarkers and to delineate CAR T-based therapeutic strategies targeting the immunopathologic cascade, aimed to enhance anti-cancer activity and confine toxicity. Keywords: Toxicity, CAR-T, B cell lymphoma, Immunophenotype